Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Based on Ames Tets on Ethyl paraben and read across from methyl and proypl paraben

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-14 to 2012-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction (activity of metabolic activation system was validated in Ames tests using 2-aminoanthracene and benzo[a]pyrene)
Test concentrations with justification for top dose:
Experiment I (plate incorporation):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II (preincubation):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 111102, 111201, 111219
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, Applichem Lot No. 1P008076
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
-S9: 10 µg/plate sodium azide in Aqua dest. (TA100, TA1535); 4-nitro-o-phenylene-diamine in DMSO (10 µg/plate for TA98, 40 µg/plate for TA1537); 1 µL/plate methylmethanesulfonate in Aqua dest. (TA102); +S9: 2-aminoanthracene in DMSO for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION:
experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates each in two independent experiments were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 1537 at a concentration of 5000 µg/plate (with and without metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 102 toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with and without metabolic activation).

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Ethylparaben did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Ethylparaben is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Ethylparaben at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

Ethylparaben was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep-2018 to Feb-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted on 29 th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and ß-Naphthoflavone induced rat liver homogenate
Test concentrations with justification for top dose:
The test item was found soluble in DMSO at 200 mg/mL. Since the test item showed slight precipitation and no changes in pH up to 2 mg /mL, same concentration was considered as the highest concentration in the initial cytotoxicity test.
There was no excessive cytotoxicity up to 2 mg/mL, therefore 2 mg/mL was selcted as the highest concentration for testing in the gene mutation test.
For concentrations i.e. 0.25, 0.50, 1 and 2 mg/ml were selcted for gene mutation test based on initial cytotoxicity test.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
The derivative of the CHO-K1, CHO AA8 cells were used as the test system as recommended in teh OECD TG 476. CHO AA8 cells, Batch No. 5000062 procured fromAmerican Type Culture Collection (ATCC) was used for the test.

Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test.

Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells, according to OECD TG 476 " In vitro Mammalian cell genen mutation test using hprt and xprt genes" adopted on 29th july 2016.

The test item was found soluble in DMSO at 200 mg/mL. Slight precipitation was observed at 2 mg/ml. No change in pH and precipitate at and up to 2 mg/mL in culure medium. Based on the result of solubility, pH and precipitation test an initial cytotoxicity test was conducted at concentrations of 0.0625, 0.125, 0.25, 0.5 1 and 2 mg/mL using DMSO as vehicle in the presence and absence of metabolic activation (3 to 6 hours). The result of the initial cytotoxicity test indicated that the test item was not cytotoxic to CHO AA8 cells, as the relative survival of the treated CHO AA8 cellsat the test concentrations up to 2 mg/mL was in a range of 71.91 to 86.21 % when compared with the respective vehicle control, both in the presence and absence of metabolic activation. The gene mutation test was conducted at concentrations of 0.25, 0.5, 1 nd 2 mg/mL in for plates/groups in the presence and absence of metabolic activation (3 to 6 hours) Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test. Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.

There was no statistically significant increase in number of mutant colonies at any of concentrations tested when compared with the vehicle control. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control.

Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read across justfication (chapter 13)
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Results of Propyl paraben (Source substance)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Results of Methyl paraben source substance
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-16 to 2012-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I / without metabolic activation / 4 hours treatment:
7.0, 14.0, 28.0, 56.0, 93.0, 112.0 µg/mL

Experiment I / with metabolic activation / 4 hours treatment:
28.0, 56.0, 112.0, 224.0, 336.0, 448.0 µg/mL

Experiment II / without metabolic activation / 24 hours treatement:
14.0, 28.0, 56.0, 112.0, 168.0, 224.0 µg/mL

Experiment II / with metabolic activation / 4 hours treatment:
28.0, 56.0, 112.0, 224.0, 336.0, 448.0 µg/mL

In experiment I with and without metabolic activation and in experiment II with metabolic activation the cultures at the maximum concentration were not continued due to exceed-ingly severe cytotoxicity. In the second experiment without metabolic activation the cultures at the two highest concentrations were not continued for the same reason.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.35 measured in the solvent control versus pH 7.34 measured at 1800 µg test item/mL
- Effects of osmolality: Not increased (383 in the solvent control versus 362 measured at 1800 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation: In the main experiments I and II no precipitation occurred.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 14.1 and 1800 µg/mL (≈ 10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50% were noted at 112.5 µg/mL and above without metabolic activation following 4 hours treatment. In the presence of metabolic activation cytotoxic effects were observed at 450 µg/mL and above. After 24 hours treatment the cell growth was completely inhibited at 225 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treat-ment period prior to removal to the test item. Precipitation occurred at 900 µg/mL in the pres-ence and absence of metabolic activation following 4 and 24 hours treatment.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
      % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12
Experiment I / 4 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 10.1 1.0 100.0 100.0 100.0 20.7 1.0
Positive control (EMS) 150.0 - 99.2 90.3 80.1 145.3 14.4 95.1 101.2 94.4 179.5 8.7
Test item 7.0 - 108.9 107.0 106.0 9.5 0.9 98.8 87.3 107.9 24.0 1.2
Test item 14.0 - 100.2 94.0 113.4 11.5 1.1 102.5 86.5 120.5 6.2 0.3
Test item 28.0 - 101.7 108.1 96.6 27.3 2.7 97.5 83.4 114.1 10.5 0.5
Test item 56.0 - 77.9 82.9 113.9 6.3 0.6 74.0 71.6 106.8 2.5 0.1
Test item 93.0 - 18.1 26.7 116.6 6.0 0.6 20.4 22.3 101.8 2.2 0.1
Test item 112.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Solvent control with DMSO + 100.0 100.0 100.0 14.0 1.0 100.0 100.0 100.0 11.8 1.0
Positive control (DMBA) 1.1 + 24.1 93.3 87.7 1105.5 79.2 25.7 74.5 85.4 751.9 63.6
Test item 28.0 + 82.5 133.1 90.6 12.4 0.9 89.5 118.4 107.2 9.3 0.8
Test item 56.0 + 95.2 125.9 98.5 14.2 1.0 100.8 103.9 106.9 11.2 0.9
Test item 112.0 + 86.9 130.6 95.8 13.2 0.9 94.4 103.4 94.8 7.3 0.6
Test item 224.0 + 79.3 118.9 100.8 4.9 0.4 97.1 90.8 93.4 4.2 0.4
Test item 336.0 + 14.3 117.8 95.7 7.8 0.6 13.0 95.1 100.7 6.8 0.6
Test item 448.0 + 0.2 culture was not continued# 0.3 culture was not continued#
Experiment II / 24 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 15.8 1.0 100.0 100.0 100.0 9.5 1.0
Positive control (EMS) 150.0 - 107.9 86.8 89.2 315.0 19.9 98.1 87.3 85.6 309.2 32.5
Test item 14.0 - 88.3 89.9 102.5 15.3 1.0 90.8 107.2 95.5 16.6 1.7
Test item 28.0 - 86.8 78.0 98.5 0.0 0.0 98.2 90.5 102.9 13.7 1.4
Test item 56.0 - 90.7 77.5 92.9 9.0 0.6 78.8 76.1 91.9 16.8 1.8
Test item 112.0 - 75.0 51.9 88.2 18.1 1.1 67.1 50.4 93.7 21.3 2.2
Test item 168.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Test item 224.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Experiment II / 4 h treatment        
Solvent control with DMSO + 100.0 100.0 100.0 18.5 1.0 100.0 100.0 100.0 22.0 1.0
Positive control (DMBA) 1.1 + 29.6 65.4 68.4 987.0 53.4 39.5 56.9 74.3 1056.5 48.1
Test item 28.0 + 102.3 115.9 89.9 6.5 0.4 92.2 92.0 75.5 16.7 0.8
Test item 56.0 + 103.2 115.5 78.1 15.6 0.8 101.7 101.3 121.6 10.9 0.5
Test item 112.0 + 101.8 126.2 90.8 23.4 1.3 98.2 96.8 127.8 11.0 0.5
Test item 224.0 + 100.5 103.0 99.5 17.5 0.9 96.8 91.0 93.5 14.8 0.7
Test item 336.0 + 24.6 66.3 97.8 21.7 1.2 17.3 35.9 99.1 3.2 0.1
Test item 448.0 + 0.0 culture was not continued# 0.0 culture was not continued#

# culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:
Interpretation of results:
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, NIPASOL M (= Propylparaben) is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part without metabolic activation was prematurely terminated due to microbial contamination. This part of the experiment was repeated as experiment IA. The data of experiment IA are reported as experiment I without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted in the first experiment at 336.0 µg/mL with and at 93.0 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described above occurred at 336.0 µg/mL with metabolic activation. In the experimental part of the second experiment without metabolic activation a very steep cytotoxic gradient was observed. Borderline cytotoxicity was noted at 112.0 µg/mL (relative cell density of 51.9 and 50.4%). Evaluation of any data on mutagenicity was impossible at the next higher concentration of 168.0 µg/mL as exceedingly severe cytotoxic effects completely inhibited the cell growth. The recommended cytotoxic range of approximately 10- 20% relative cloning efficiency I or relative cell density

was covered with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained within the historical range of solvent controls. The induction factor did not reach or exceed the threshold of 3 time the mutation frequency of the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations.In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.5 up to 22.0 mutants per 106 cells; the range of the groups treated with the test item was from 0.0 up to 27.3 mutant colonies per 106 cells. EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 2018 to 07 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: external sample from China
- Expiration date of the batch: 27.10.2019
- Purity test date:99,7%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle:Dimethyl sulphoxide
Target gene:
Human lymphocytes
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human
- Cell cycle length, doubling time or proliferation index: 44 to 48 hours
- Sex, age and number of blood donors if applicable: male and female donors (21 and 33 years of age)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes:46±2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640, 5 PERCENT
- Properly maintained: yes
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
1 mL of S9 homogenate was mixed with 9 mL co factor
Test concentrations with justification for top dose:
As no cytotoxicity or precipitation observed, the highest test concentration correspond to 2 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item was found soluble in dimethyl sulphoxide at 200 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 to 6 hours and 20 to 22 hours


STAIN (for cytogenetic assays): Acridine orange

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellet were mixed with 3 mL of freshly prepared 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 2000 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3) and slides were prepared.
Clean slides were stored in a container with distilled and kept in the refrigerator for 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension was aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment/group and slide number. The slides were air dried. Smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes

NUMBER OF CELLS EVALUATED: 1000 per replicate


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronucleus in binucleates

DETERMINATION OF CYTOTOXICITY
- Method: Measurement of the relative frequencies of mononucleate, binucleate and multi-nucleate cells in the culture was made to determine cell proliferation and the cytostatic activity
Evaluation criteria:
Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures was determined from each treatment.
The CBPI was calculated for all treated and control cultures as measurement of cell cycle delay.
Slides was evaluated for the presence of Micronucleus in at least 2000 Binucleates (at least 1000 per culture).
Statistics:
Data was analyzed using SPSS software version 22 for differences among vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p≤0.05) and the statistical significance was designated by the superscripts thoughout the study report as stated below.
*: Statistically significant (p≤0.05).
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- pH: no change
- Water solubility: insolublle
- Precipitation: no


RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used) : Cytochalasin B
- Distribution of mono-, bi- and multi-nucleated cells: yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000 per culture
- Indication whether binucleate or mononucleate where appropriate: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:CBPI
Remarks on result:
other: .
Remarks:
Results are summerized in the next part

TABLE 1.      SUMMARY OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment

Concentration

mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.62

0.0

2039

3

0.15

2

Propylparaben

0.5

1

1.58

6.45

2099

4

0.19

2

1

1

1.56

9.68

2030

3

0.15

2

2

1

1.54

12.90

2035

5

0.25

2

Cyclophosphamide Monohydrate

10 µg/mL

1

1.61

1.61

2007

26

1.30*

2

TABLE 2.      SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2016

3

0.15

2

Propylparaben

0.5

1

1.61

3.17

2015

3

0.15

2

1

1

1.57

9.52

2071

3

0.15

2

2

1

1.54

14.29

2020

2

0.10

2

Colchicine

0.03 µg/mL

1

1.61

3.17

2053

26

1.27*

2

Table 3 SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 20 TO 24 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2031

3

0.15

2

Propylparaben

0.5

1

1.59

6.35

2031

3

0.15

2

1

1

1.54

14.29

2018

3

0.15

2

2

1

1.44

30.16

2060

4

0.19

2

Mitomycin C

0.075 µg/mL

1

1.60

4.76

2077

26

1.25*

2

Conclusions:
Based on the results obtained in this study, it is concluded that the test item Propylparaben is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 2 mg/mL both in short term and long term treatment, both in presence and absence of metabolic activation as it showed no evidence of increase in the induction of micronuclei under the test conditions.
Executive summary:

The test item was evaluated for the formation of micronuclei in the cytoplasm of interphase cells according to OECD TG 487  “In vitro Mammalian Cell Micronucleus Test” .Test item was found soluble in dimethyl sulphoxide at 200 mg/mL. Moderate and mild precipitation was observed at 2 and 1 mg/mL respectively. No precipitation was observed in any other concentration tested. No change in pH was observed in any of the concentrations tested. Hence, 2 mg/mL was selected as highest concentration for initial cytotoxicity test and other concentrations tested were 0.125, 0.25, 0.5, 1 and 2 mg/mL using DMSO as a vehicle. The experiment was conducted with and without metabolic activation. The test item was assessed in proliferated lymphocytes in duplicates by exposing for a short term (3 to 6 hours, with and without metabolic activation) and a long term (20 to 24 hours, without metabolic activation).

Cytokinesis was blocked using Cytochalasin B, the cells were harvested and slides were prepared. In order to assess the cytotoxicity of the test item, the Cytokinesis-Block Proliferation Index (CBPI) was calculated for cultures treated with the test item and vehicle control.  

In initial cytotoxicity test, the test item at the concentrations of 0.125, 0.25, 0.50, 1 and 2 mg/mL resulted in a percentage reduction of average Cytokinesis-Block Proliferation Index (CBPI) was in the range of 1.67 to 30.00 at the tested concentrations in both short term and long term treatment. The percentage reduction of average Cytokinesis-Block Proliferation Index (CBPI) was not greater than 45±5% at 2 mg/mL. Hence 2 mg/mL was selected as highest concentration for the micronucleus test. Other concentrations tested were 0.5 and 1 mg/mL.

In micronucleus test, the test item was tested at the concentrations of 0.5, 1 and 2 mg/mL for short term in presence and absence of metabolic activation and long term treatment in the absence of metabolic activation system. The test item did not induced cytotoxicity up to 2 mg/mL when compared to vehicle control. No statistical significant increase in the percentage of micronuclei in binucleated cells observed in any of the tested concentration when compared to the vehicle control. The positive controls resulted increase in the micronuclei frequency with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of test system towards positive control and confirmed that the test conditions were adequate and within the range of historical control.

Conclusion

Based on the results obtained, it is concluded that the test item Propylparaben is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 2 mg/mL both in short term and long term treatment, both in presence and absence of metabolic activation as it showed no evidence of increase in the induction of micronuclei under the test conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification (chapter 13)
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: See provided data on propyl paraben
Remarks:
non mutagenic

TABLE 1.      SUMMARY OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS for the read-across substance propyl paraben

Treatment

Concentration

mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.62

0.0

2039

3

0.15

2

Propylparaben

0.5

1

1.58

6.45

2099

4

0.19

2

1

1

1.56

9.68

2030

3

0.15

2

2

1

1.54

12.90

2035

5

0.25

2

Cyclophosphamide Monohydrate

10 µg/mL

1

1.61

1.61

2007

26

1.30*

2

TABLE 2.      SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2016

3

0.15

2

Propylparaben

0.5

1

1.61

3.17

2015

3

0.15

2

1

1

1.57

9.52

2071

3

0.15

2

2

1

1.54

14.29

2020

2

0.10

2

Colchicine

0.03 µg/mL

1

1.61

3.17

2053

26

1.27*

2

Table 3 SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 20 TO 24 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2031

3

0.15

2

Propylparaben

0.5

1

1.59

6.35

2031

3

0.15

2

1

1

1.54

14.29

2018

3

0.15

2

2

1

1.44

30.16

2060

4

0.19

2

Mitomycin C

0.075 µg/mL

1

1.60

4.76

2077

26

1.25*

2

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
accepted: 15. Sept. 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Important aspects of study design (with/without metabolic activation, 3 hours incubation time, 24 hours recovery ect.) are in line with the current OECd guideline, but study is restricted because the testing of only one concentration and due to the moderate study reporting.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no negative control and testing only of only one concentration reported
GLP compliance:
no
Remarks:
performed before GLP guidelines
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
A clonal sub-line from the lung of a male Chinese hamster (CHL) was used. The modal chromosome number of this cell line is 25.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.125 mg/mL (125 µg/mL)
Vehicle / solvent:
Ethanol or Dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Saline, Ethanol, Dimethyl sulfoxide
True negative controls:
yes
Remarks:
Untreated cells
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
Preparation of S9 mix:
Liver microsome fraction (S9) was prepared from male Wistar rats 8 weeks of age. For induction of microsomal enzymes, rats were given: PCB, as a single i.p. injection of 500 mg/kg bw 5 days before they were killed; phenobarbital, 2 i.p. injections of 80 mg/kg bw, 2 days and 1 day before they were killed; 3-Methylcholanthrene, a single i.p. injection of 50 mg/kg bw 2 days before they were killed. S9 mix from untreated rats served as control.The livers were carefully homogenated and the homogenate adjusted to 2 mL/g of wet liver weight and then centrifuged at 0-4°C for 20 min. at 9000 x g. Supernatant was stored at -80°C until used. All these procedures were done under aseptic conditions.
The microsome reaction mixture (S9) consisted of 3 mL of S9 fraction, 2 mL of 20 mM HEPES buffer (pH 7.2), 1 mL of 50 mM MgCl2, 1 mL of 330 mM KCl, 1 mL of 50 mM Glucose 6-phosphate, 1 mL NADP and 1 mL of distilled water. The S9 mix was prepared just before use and sterilized by filtration except the S9 fraction.

Incubation procedure with S9 mix:
A cell suspension was made from 3-day-old cultures by trypsinisation, and the cell number was adjusted to 1.2 x 10exp7/mL. 0.2 mL of the suspension was mixed with 0.2 mL of Methylparaben and 1 mL of S9 mix in each test tube. The tubes were incubated at 37°C and shaken continuously for 3 hours. After centrifugation, the cells were cultured for additional 24 hours.

Chromosome observation:
Cells were treated with 0.2 µg of Colcemid for 2 hours, and after trypisinisation, incubated in 0.075 M KCl hypotonic solution for 15 min. at 37°C. The cells were fixed with ice-cold fixative (Methanol:Glacial acetic acid, 3:1) which was changed 3 times.A few drops of the cell suspension were placed on clean slides on wet blotting paper. The slides were stained with 1% Giemsa solution (pH 6.8) for 15 min.The number of cells with chromosomal aberration was counted on 100 well-spread metaphases. The inicdence of polyploid cells in the 100 metaphases was also recorded.
Evaluation criteria:
The types of aberration were classified into 5 groups:
- chromatid gaps
- chromatid breaks
- chromatid or chromosomal exchanges
- ring formation
- fragmentation or pulverisation
Breaks narrower than the width of a chromatid were grouped in gaps.
The final judgement given to all experimental groups was:
- negative, if less than 4.9% of the aberration was detected even when doses of the test item were elevated to sub-amount, where almost no mitosis was observed
- suspicious, if between 5.0 to 9.9%
- positive, if between 10 to 19.9% (+), 20 to 49.9% (++) or more than 50% (+++)
When no reasonable dose-response was obtained, additional experiments with different doses were carried out to confirm its reproducibility.
Statistics:
no data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
5 to 9.9% aberrations
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

Methylparaben did not induce chromosomal aberrations in the absence of metabolic activation but induced aberrations in the presence of metabolic activation. In this in vitro study the test substance is considered to be non-mutagenic without S9 mix and mutagenic with S9 mix.
Executive summary:

The potential of Methylparaben to induce chromosome aberrations was investigated in Chinese hamster lung cells in vitro. The test compound was tested at 125 µg/mL with and without metabolic activation. Benzo[a]pyrene as reference mutagen showed an increase in the number of chromosome aberrations.

Methylparaben did not induce chromosome aberrations in the absence of S9 mix but was positive in the presence of S9 mix.

Methylparaben is therefore considered to non-mutagenic without and slightly mutagenic with metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only short abstract available.
Principles of method if other than guideline:
Reverse mutation assay using S. typhimurium strains according to the method of Ames, McCann and Yamasaki (1975).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA92 and TA94
Metabolic activation:
with
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Fischer rats pretreated with polychlorinated biphenyls.
Test concentrations with justification for top dose:
six concentrations up to 5 mg/plate
Vehicle / solvent:
- Vehicle/solvent used: Dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA92 and TA94
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Conclusions:

negative with metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study, tested with the source substance Methylparaben. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
no
GLP compliance:
no
Remarks:
study performed before
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
10 to 12 week old, male, albino rats obtained from a closed colony (random-bred) were used.
Body weight: 280 to 350 g (400 g in case of high dose group rats)
The animals were fed a commercial 4% fat diet water ad libitum until they were put on experiment. Periodic tests to verify the absence of coliforms, Salmonella and Pseudomonas sp. were performed.
Acclimatisation period: 4 to 11 days
Housing: 5/cage
Identification: Ear punch
Sanitary cages and bedding used, and changed 2 times per week, at which time water containers were cleaned, sanitised and filled. Once a week, cages were repositioned on racks; racks were repositioned within rooms monthly.
Route of administration:
oral: gavage
Vehicle:
0.85% saline
Details on exposure:
Dose levels employed for Methylparaben:
- acute (single dose): 5, 50, 500, 5,000 mg/kg bw
- subacute (5 doses on 5 consecutive days): 5, 50, 500, 5,000 mg/kg bw
Duration of treatment / exposure:
n.a. (gavage study)
Frequency of treatment:
acute: 1 single oral administration
subacute: once a day for 5 consecutive days (24 hours apart)
Post exposure period:
8 weeks (sequential matings)
Remarks:
Doses / Concentrations:
5
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
50
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
500
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
5000
Basis:
actual ingested
mg/kg body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
0.3 mg/kg bw Triethylene Melamine intraperitoneally
Tissues and cell types examined:
determination of fertility index
Necropsy of the uteri of mated females
- early deaths (deciduomata)
- absorptions
- dead implatations
- total implantations
- Number of Corpora lutea
Details of tissue and slide preparation:
Following treatment, the males were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Females were killed using CO2 at 14 days after separating from the male, and at necropsy the uterus was examined for deciduodimata, late fetal deaths and total implantations.
Corpora lutea, early fetal deaths, late fetal deaths and total implantations per uterine horn were recorded.
Evaluation criteria:
Each male was mated with 2 females per week, and this provided for an adequate number of implantations per group per week (200 minimum) for negative controls, even if there was a 4-fold reduction in fertility of implantations.
Statistics:
yes, please refer to "any other information..." below
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
100 and 500 mg/kg: no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity:
Test I
1000 mg/kg: 1 of 5 animals dead; reddened stomach lining, lung congested (day 3)
2000 mg/kg: 2 of 5 animals dead, reddened stomach lining, lung congested (day 2)
3000 mg/kg: 4 of 5 animals dead, reddened stomach lining, lung congested (day 1: 3 animals, day 2: 1 animal)
4000 mg/kg: 4 of 5 animals dead, reddened stomach lining, lung congested (day 1: 4 animals)
5000 mg/kg: 10 of 10 animals dead, reddened stomach lining, lung congested (day 1: 10 animals)
=> LD50 2,100 mg/kg (Litchfield-Wilcoxon method)

Test II
A single dose of 5000 mg/kg bw Methylparaben was administered to 10 male rats (average body weight: 262 g). No signs of toxicity or abnormal behavior were observed in the 7-day observation period. No deaths occured. At termination all animals were killed and on necropsy no gross findings were observed.
=> LD50 > 5000 mg/kg
Conclusions:
Interpretation of results (migrated information): negative
Methylparaben is considered to be non-mutagenic in rats in the Dominant Lethal Assy applying dosages up to 5000 mg/kg bw (at single dose or 5 dosages on 5 consecutive days).
Executive summary:

Methylparaben was tested in the dominant lethal assay in rats. The test item was suspended in 0.85% saline and dosed at 5, 50, 500 and 5000 mg/kg bodyweight to male rats (acute: single dose; subacute: 5 doses at 5 consecutive days), upon the results of the previously conducted dose range finding study. According to the test procedure the animals were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Females were killed at 14 days after mating and at necropsy the uterus was examined for the number of Corpora lutea, early deaths, late fetal deaths and total implantations.

Triethylene Melamine (TEM) was used as positive control substance and administered intraperitoneally at a dose of 0.3 mg/kg bodyweight. TEM caused significant preimplantation loss and embryo resorption during the first 5 weeks.Saline was used as negative control.

Methylparaben is considered to be non-mutagenic in rats in this dominant lethal assay when using dosages of 5, 50, 500 and 5000 mg/kg bw/d since no dose response or time trend patterns, which would suggest an effect, were observed.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read across justification (chapter 13)
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
100 and 500 mg/kg: no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The in vitro genetic toxicity of ethylparaben was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471 and GLP (Donath, 2012). Two independent experiments (plate incorporation and preincubation) were conducted with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 at concentrations up to 5000 µg/plate with and without metabolic activation. The test substance did not induce reversions in any of the strains tested with or without metabolic activation. The used positive controls were valid. In the plate incorporation assay, cytotoxicity was observed in tester strains TA 98 and TA 1537 at 5000 µg/plate and in TA 1535, TA 100 and TA 102 at concentrations of above 2500 µg/plate. After preincubation, cytotoxicity was noted in tester strains TA 100, TA 1535 and TA 1537 at concentrations of above 1000 µg/plate and in TA 98 and TA 102 at concentrations of above 2500 µg/plate. No precipitation of the test substance was observed in any experiment up to the highest dose tested.

 

In a further bacterial reverse mutation assay (Ames test) the genetic toxicity of ethylparaben was determined in tester strains TA 1535, TA 1537, TA 98, TA 100, TA 92 and TA 94 with metabolic activation (Ishidate, 1984). After preincubation with concentrations up to 5000 µg/plate, the test substance was not genotoxic in any tester strain.

 

There are no further data available on the genetic toxicity of ethylparaben. However, there are reliable data for methylparaben and propylparaben which are structurally related to ethylparaben.Therefore, read-across was performed based on an analogue approach. For a detailed justification of the analogue approach, please refer to section 13 of the technical dossier. The target substance and the source substances form a homologue series of esters of p-hydroxybenzoic acid and differonly in the length of the alkyl side chain, which contains 1, 2 or 3 carbon atoms for methylparaben, ethylparaben and propylparaben, respectively.

The toxicokinetic behaviour of the target and the source substances is comparable. The substances were shown to be rapidly absorbed, metabolised and excreted mainly via urine. The main metabolites of the three substances were p-hydroxybenzoic acid, p-hydroxyhippuric acid and their glucuronic acid conjugates.

Available data on ethylparaben did not show reactive properties in bacterial mutation studies (Ames test) and no genotoxic effects were observed for propylparaben in several in vitro studies, i.e. Ames test and HPRT test as well as for methylparaben in vivo. Additionally, no DNA binding alerts were found for the target and the source substances by OECD Toolbox profiling. It is considered that there is no evidence for generation of chemically reactive metabolites for the target and the source substances.

In conclusion, as methylparaben, ethylparaben and propylparaben were shown to have comparable toxicological properties, it is considered appropriate to read-across from methylparaben and propylparaben to ethylparaben.

 

The induction of gene mutations in mammalian cells by propylparaben was assessed in a HPRT test according to OECD 476 and GLP (Wollny, 2012). The test was conducted in two independent experiments in V79 cells with metabolic activation at concentrations up to 448 µg/mL for 4 h and without metabolic activation at concentrations up to 224 µg/mL for 4 and 24 h. Cytotoxic effects were observed in both experiments at concentrations of above 93 µg/mL without metabolic activation and at concentrations of above 336 µg/mL with metabolic activation. No genotoxicity was observed up to the highest concentrations tested. The used positive controls were valid.

 

Methylparaben was tested in a dominant-lethal test in rats according to OECD 478 (Litton Bionetics Inc, 1974). Methylparaben was applied to male rats in doses up to 5000 mg/kg bw/d by oral gavage in a single treatment and daily on 5 consecutive days. During the post exposure period (8 week for the single treatment group and 7 weeks for the subacute group), the male rats were sequentially mated to 2 females per week. Two weeks after mating, the females were sacrificed and at necropsy the uteri were examined for corpora lutea, early deaths, late fetal deaths and number of total implantations. No dose response relationship or time trend pattern was observed across the groups. Therefore, methylparaben was considered to be non-mutagenic in rats in the dominant-lethal assay.

 

In an in vivo Mammalian Bone Marrow Chromosome Aberration test performed in male rats according to OECD 475, methylparaben did not induce chromosome aberrations up to 500 mg/kg bw/d (Litton Bionetics Inc, 1974).


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. The available studies on in vitro gene mutation in bacteria, in vitro gene mutation in mammalian cells and in vivo chromosome aberration were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Bacterial reverse mutation assay, Ames test (OECD 471): negative
Mammalian Cell Gene Mutation Test, HPRT test (OECD 476): negative
Rodent Dominant Lethal Test (OECD 478): negative
Mammalian Bone Marrow Chromosome Aberration Test (OECD 475): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance and on surrogate substances does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.